WO2023147239A1 - Synthèse enzymatique de polynucléotide - Google Patents

Synthèse enzymatique de polynucléotide Download PDF

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Publication number
WO2023147239A1
WO2023147239A1 PCT/US2023/060847 US2023060847W WO2023147239A1 WO 2023147239 A1 WO2023147239 A1 WO 2023147239A1 US 2023060847 W US2023060847 W US 2023060847W WO 2023147239 A1 WO2023147239 A1 WO 2023147239A1
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endonuclease
nucleotide
nucleic acid
guiding
endo
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PCT/US2023/060847
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English (en)
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Cheng-Yao Chen
Yi-Wen Cheng
Tsu-Ying WU
Wen-Ting Chen
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Chen cheng yao
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present disclosure relates to a method for enzymatic synthesis of nucleic acids or polynucleotides under various conditions, and a kit for implementation of such method.
  • the enzymatic nucleic acid synthesis approach relies on a templateindependent polymerase, such as terminal deoxynucleotidyl transferase (TdT) to repeatedly add nucleotides to an initiator.
  • TdT terminal deoxynucleotidyl transferase
  • the initiator which is normally composed of a single-stranded nucleic acid, or polynucleotide, serves as a starting point for synthesizing new nucleic acids or polynucleotide.
  • the synthesis initiator needs to be immobilized on a solid support.
  • the enzymatic nucleic acid synthesis can continue to elongate the nucleic acid strand or polynucleotide chain from the initiator until the desired length and sequence are obtained.
  • US Patent No. 10,683,536 B2 describes an enzymatic method for synthesizing polynucleotides, which requires a specific cleavage agent, such as an alkaline solution, a metal ion, and a type II restriction endonuclease, to decouple the newly synthesized nucleic acid or polynucleotide from the nucleic acid initiator.
  • a specific cleavage agent such as an alkaline solution, a metal ion, and a type II restriction endonuclease
  • nascent nucleic acid or polynucleotide utilizing the alkaline solution requires a unique, chemical linker between the newly synthesized nucleic acid and the initiator for the site-specific cleavage reaction, which is inconvenient for routine implementation.
  • the disclosure regarding the cleavage of nascent nucleic acid or polynucleotide from an initiator using the type II restriction endonuclease still lack examples to prove the feasibility.
  • the type II restriction endonuclease requires a sequence-specific recognition site, such as a palindromic sequence typically with 4 to 8 bases, for DNA strand cleavage, which limits its broad adaptations in de novo enzymatic nucleic acid synthesis.
  • US Patent Application Publication No. 2021/0254114 Al describes a method for cleaving a polynucleotide product of template-free enzymatic nucleic acid synthesis, in which an initiator is devised to harbor a site-specific 3 ’-penultimate deoxyinosine (di).
  • the position of di in the initiator can be recognized by endonuclease V (EndoV) and served as a guide for EndoV to precisely cleave the newly synthesized DNA strand from the initiator.
  • EndoV endonuclease V
  • the DNA-strand cleavage activity is solely achieved by E.
  • Provided herein discloses a method and a kit of preparing synthetic polynucleotides for multiple applications, for example, for the preparation of custom polynucleotides, DNA or RNA probes.
  • a site-specific retrieving method and kit to obtain predetermined sequence and length of polynucleotides according to user’s intention.
  • the user may configure the sequence and length of the predetermined polynucleotides as well as the position of a guiding nucleotide in the newly synthesized nucleic acids and directly retrieve the desired predetermined polynucleotides by a site-specific polynucleotide cleavage using a single enzyme. Therefore, the method and a kit disclosed herein enable the user to obtain the predetermined sequence and length of polynucleotide precisely and efficiently.
  • the method provided herein is a method of synthesizing a polynucleotide enzymatically, the method comprises: providing an initiator comprising a 3 ’-terminal nucleotide having a free 3 ’-hydroxyl group; incorporating nucleotide monomers to the initiator by a polymerase to elongate a nucleic acid strand from the free 3 ’-hydroxyl group, wherein the nucleotide monomers comprise a guiding nucleotide to be recognized by an endonuclease, such that the guiding nucleotide is incorporated at a specific position of the newly synthesized nucleic acid strand; and subjecting the endonuclease to cleave the nucleic acid strand according to a position of the guiding nucleotide to release a predetermined sequence and length of polynucleotide and leaves a remaining nucleic acid strand or the initiator having a free 3 ’-hydroxy group, wherein the
  • the method provided herein is used in a templateindependent or a template-dependent (i.e., templated-directed) polynucleotide synthesis.
  • the initiator is a single-stranded nucleic acid having a free 3 ’ hydroxyl group for elongating a nascent nucleic acid strand.
  • the initiator is a single-stranded primer annealed with a complimentary template to form a primer-template duplex for directing the nucleic acid synthesis by polymerase. The polymerase elongates the primer according to the sequence information of the template.
  • the guiding nucleotide is a natural, unnatural, or modified nucleotide, wherein the nucleobase of the nucleotide may be, for example, an uracil, a xanthine, or a hypoxanthine.
  • the guiding nucleotide is a natural, unnatural, or modified nucleotide, such as nucleotide containing an uracil or inosine.
  • the method provided herein comprises the use of a template- independent or a template-dependent polymerase, wherein the template independent polymerase may be, for example, a B-family DNA polymerase.
  • the endonuclease derives from Thermococcus barophilus (Tba), Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac), Bacillus pumilus (Bpu), Pyrococcus abyssi (Pab), Thermococcus kodakarensis (Tko), Thermococcus gammatolerans (Tga), or Bacillus subtilis (Bsu).
  • the method provided herein comprises use of a hyperthermophilic or mesophilic endonuclease capable of specifically recognizing an incorporated guiding nucleotide in a newly synthesized nucleic acid strand or polynucleotide chain, therefore the endonuclease and cleave the nucleic acid strand or polynucleotide chain according to the position of guiding nucleotide in the nucleic acid strand or polynucleotide chain, wherein the second phosphodiester bond 3’ to the guiding nucleotide, the first phosphodiester bond 5' to the guiding nucleotide, the second phosphodiester bond 5' to the guiding nucleotide, or the third phosphodiester bond 5' to the guiding nucleotide can be specifically cleaved by the corresponding endonuclease, respectively.
  • the hyperthermophilic endonuclease derives from the group consisting of Thermococcus barophilus endonuclease V (Tba Endo V), Pyrococcus furiosus endonuclease V (Pfu Endo V), Thermococcus kodakarensis endonuclease V (Tko Endo V), Pyrococcus furiosus endonuclease Q (Pfu Endo Q), Methanosarcina acetivorans endonuclease Q (Mac Endo Q), Pyrococcus abyssi NucS endonuclease (Pab NucS), Thermococcus kodakarensis EndoMS endonuclease (Tko EndoMS), Thermococcus gammatolerans NucS endonuclease (Tga NucS) and the enzyme variants or mutants
  • the method provided herein comprises use of a mesophilic endonuclease from the group such as Bacillus subtilis endonuclease V (Bsu Endo V; SEQ ID NO: 11), Bacillus pumilus endonuclease Q (Bpu Endo Q; SEQ ID NO: 12), and the enzyme variants thereof in the newly synthesized nucleic acid strand or polynucleotide chain.
  • a mesophilic endonuclease from the group such as Bacillus subtilis endonuclease V (Bsu Endo V; SEQ ID NO: 11), Bacillus pumilus endonuclease Q (Bpu Endo Q; SEQ ID NO: 12), and the enzyme variants thereof in the newly synthesized nucleic acid strand or polynucleotide chain.
  • the endonuclease derives from the group consisting of Bacillus subtilis endonuclease V (Bsu Endo V), Escherichia coli endonuclease V (Eco Endo V; SEQ ID NO: 7), Pyrococcus furiosus endonuclease V (Pfu Endo V; SEQ ID NO: 8), Thermococcus barophilus endonuclease V (Tba Endo V; SEQ ID NO: 10), Thermococcus kodakarensis endonuclease V (Tko Endo V; SEQ ID NO: 16) or the enzyme variants thereof and specifically cleaves at the second phosphodiester bond 3’ to the guiding nucleotide.
  • the endonuclease is selected from the group consisting of Pyrococcus furiosus endonuclease Q (Pfu Endo Q; SEQ ID NO: 9), Methanosarcina acetivorans endonuclease Q (Mac Endo Q; SEQ ID NO: 14), Bacillus pumilus endonuclease Q (Bpu Endo Q; SEQ ID NO: 12) and the enzyme variants thereof and specifically cleaves at the first phosphodiester bond 5’ to the guiding nucleotide in the newly synthesized nucleic acid strand or polynucleotide chain.
  • the endonuclease derives from the group consisting of Thermococcus gammatolerans NucS endonuclease (Tga NucS; SEQ ID NO: 15), Pyrococcus abyssi NucS endonuclease (Pab NucS; SEQ ID NO: 13) and the enzyme variants thereof and specifically cleaves at the second phosphodiester bond 5’ to the guiding nucleotide in the newly synthesized nucleic acid strand or polynucleotide chain.
  • Tga NucS Thermococcus gammatolerans NucS endonuclease
  • Pab NucS Pyrococcus abyssi NucS endonuclease
  • the enzyme variants thereof specifically cleaves at the second phosphodiester bond 5’ to the guiding nucleotide in the newly synthesized nucleic acid strand or polynucleotide chain.
  • the endonuclease is selected from the group consisting of Thermococcus kodakarensis EndoMS endonuclease (Tko EndoMS; SEQ ID NO: 17) and the enzyme variants thereof and specifically cleaves at the third phosphodiester bond 5’ to the guiding nucleotide in the newly synthesized nucleic acid strand or polynucleotide chain.
  • the endonuclease recognizes a guiding nucleotide in the nascent nucleic acid strand or the synthesized polynucleotide to cleave at specific phosphodiester bond near the position of guiding nucleotide.
  • the method provided herein may be implemented at higher temperatures compared to those conventionally used.
  • the temperature range for implementation can be, for example, from 10°C to 100°C, from 50°C to 90°C, or from 70°C to 90°C.
  • the nucleic acid initiator, or the synthesized/synthetic polynucleotide in a free form or duplex formation of the method provided herein is attached to a solid support and immobilized via its 5’ end.
  • the solid support can be, for example, a particle, a resin, a bead, a slide, a chip, an array, a membrane, a matrix, a flow cell, a well, a chamber, a microfluidic chamber, a channel, a microfluidic channel, a gel, a synthetic polymer, or any surface that can be attached with a synthetic nucleic acid strand or polynucleotide.
  • the present disclosure further provides a kit for synthesizing a polynucleotide enzymatically.
  • the sequence and length of polynucleotide is predetermined or designed.
  • the kit for synthesizing predetermined sequence and length of oligonucleotides of the present disclosure may comprise: a nucleic acid initiator comprising a 3 ’-terminal nucleotide having a free 3 ’-hydroxyl group; a polymerase for incorporating a plurality of nucleotide monomers to the initiator to elongate a nucleic acid strand from the free 3 ’-hydroxyl group of the initiator; wherein nucleotide monomers comprises a guiding nucleotide to be specifically recognized by an endonuclease, such that the guiding nucleotide is incorporated in the defined position of nucleic acid strand; and the endonuclease cleaves the nucleic acid strand according to the position of the guiding nucleot
  • the present disclosure further provides a kit for retrieving a predetermined sequence and length of polynucleotide enzymatically.
  • the targeting polynucleotide to be cleaved is any synthetic polynucleotide prepared in advance.
  • the kit comprises the synthetic polynucleotide, which has a site-specific guiding nucleotide; and an endonuclease for cleaving the synthetic polynucleotide to release a predetermined polynucleotide, wherein the endonuclease recognizes the position of the guiding nucleotide in the polynucleotide and cleaves at the second phosphodiester bond 3’ to the guiding nucleotide, the first phosphodiester bond 5' to the guiding nucleotide, the second phosphodiester bond 5' to the guiding nucleotide, or the third phosphodiester bond 5' to the guiding nucleotide, respectively, so that the desired predetermined polynucleotide is obtained.
  • the guiding nucleotide containing an uracil or inosine in the nucleic acid strand or a polynucleotide chain is either pre-existing or newly synthesized chemically or enzymatically.
  • FIG. 1 is a schematic diagram in accordance with at least one embodiment of the present disclosure, illustrating a de novo template-independent single-stranded DNA (ssDNA) synthesis from an initiator having a free 3 ’-hydroxyl group at 3’ end thereof, the cleavage of the newly synthesized DNA strand to obtain a desired sequence and length of polynucleotide, and the regeneration of a new free 3 ’-hydroxyl group of the remaining nucleic acid strand after cleavage of the synthesized DNA strand to obtain a predetermined polynucleotide and to serve as a new reusable initiator for the next round of nucleic acid synthesis.
  • ssDNA de novo template-independent single-stranded DNA
  • the 5’ end of the initiator is immobilized to the solid support (SS) prior to the nucleic acid synthesis reaction.
  • X represents the guiding nucleotide and is used as the origin to number nucleotide position in the nucleic acid strand.
  • N stands for an incorporated nucleotide monomer, in which the subscript numbers in ascending order (e.g., Ni, N2, N3, N4, and Ns) to the 3’ end or downstream of the strand and the subscript numbers in descending order (e.g., N-i, N-2, and N-3) to the 5’ end or upstream of the strand.
  • Ei to E4 stand for different endonucleases.
  • the scheme depicts an enzymatic nucleic acid synthetic comprising an enzymatic cleavage that may be conducted by Ei, E2, E3, or E4 at the phosphodi ester linkage between N2 and Ni, X and N-i, N-i and N-2, or N-2 and N-3, respectively, wherein the 5’ end of the initiator is attached to the solid support (SS).
  • SS solid support
  • FIGs. 2 A and 2B are schematic diagrams in accordance with at least one embodiment of the present disclosure, illustrating template-directed DNA synthesis from an initiator (i.e., primer) having a free 3’-hydroxyl group at 3’ end thereof, ; the cleavage of the nascent DNA strand to obtain a desired sequence and length of polynucleotide and the regeneration of a free 3 ’-hydroxyl group at 3’ end of the remaining nucleic acid strand to form a new reusable initiator for the next round of nucleic acid synthesis.
  • the 5’ end of the initiator is immobilized to the solid support (SS) prior to the nucleic acid synthesis reaction.
  • SS solid support
  • the template DNA is attached to the solid support via the 3 ’-end (FIG. 2A) or partially hybridized to the initiator by annealing method or other known hybridization techniques (FIG. 2B).
  • X represents the guiding nucleotide and is used as the origin to number the nucleic acid strand.
  • N stands for an incorporated nucleotide monomer, in which the subscript numbers in ascending order (e.g., Ni, N2, N3, and N4) to the 3’ end or downstream of the strand and the subscript numbers in descending order (e.g., N-i, N-2, and N-3) to the 5’ end or upstream of the strand.
  • El to E4 stand for different endonucleases.
  • the scheme depicts an enzymatic synthetic scheme comprising an enzymatic cleavage that may be conducted by Ei, E2, E3, and E4 at the phosphodiester linkage between N2 and Ni, X and N-i, N-i and N-2, or N-2 and N-3, respectively.
  • FIG. 3 is the fluorescent image of urea-polyacrylamide gel, showing the feasibility of the template-independent nucleic acid synthesis in accordance with at least one embodiment of the present disclosure.
  • Lane S refers to the initiator DNA only (Biotin- FAM-45-mer ssDNA); lane 1 refers to the initiator DNA elongated with 3’-O- (azidomethyl)-2’-deoxyuridine (U) by the polymerase; lane 2 refers to the initiator DNA elongated with 3 ’-O-(azidomethyl)-2’ -deoxy uridine (U) and 3’-O-(azidomethyl)- 2 ’-deoxy guanosine (G) consecutively, after each nucleotide-incorporation and 3’- deprotection steps; and lane 3 refers to the initiator DNA elongated with a 3’-O- (azidomethyl)-2’-deoxyuridine followed with dNTPs
  • FIGs. 4A and 4B are the fluorescent images of urea-polyacrylamide gels showing the results of Example 1 and 2, respectively, which illustrates the site-specific cleavage of the free form (FIGs. 4A (1) and 4B (1), or immobilized form (FIGs. 4A (2) and 4B (2)) of the single-stranded DNA (ssDNA), and the free form (FIGs. 4A (3) and 4B (3)) or immobilized form (FIGs. 4A (4) and 4B (4)) of the double-stranded DNA (dsDNA) by exemplary enzymes that preferentially or specifically recognize deoxyinosine (I, FIG. 4A) or deoxyuridine (U, FIG.4B), respectively, in the DNA under 37°C or 70°C.
  • deoxyinosine I, FIG. 4A
  • U deoxyuridine
  • Eco Endo V refers to the in-house prepared E. coli endonuclease V enzyme, which can be referenced to US 2021/0254114A1
  • Pfu EndoV refers to Pyrococcus furiosus endonuclease V
  • Cl refers to the commercial endonuclease V fromNew England Biolabs (NEB Eco EdoV) (Cat. #M0305S, Ipswich, MA)
  • C2 refers to the in-house developed enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and Endo VIII
  • C3 refers to an uracilspecific excision reagent from New England Biolabs (NEB USER) (Cat. #M5505S, Ipswich, MA).
  • FIGs. 5A and 5B are the fluorescent images of urea-polyacrylamide gels showing the results of Example 3 and 4, respectively, which illustrate the site-specific cleavage of the free form (FIGs. 5 A (1) and 5B (1), or immobilized form (FIGs. 5A (2) and 5B (2)) of the single-stranded DNA (ssDNA), and the free form (FIGs. 5 A (3) and 5B (3)) or immobilized form (FIGs. 5 A (4) and 5B (4)) of the double-stranded DNA (dsDNA) by the exemplary enzymes that preferentially or specifically recognize deoxyinosine (I, FIG.
  • Pfu EndoQ refers to Pyrococcus furiosus endonuclease Q
  • Cl refers to the commercial endonuclease V fromNew England Biolabs (NEB Eco EdoV) (Cat. #M0305S, Ipswich, MA)
  • C2 refers to the in-house developed enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and Endo VIII
  • C3 refers to an uracilspecific excision reagent from New England Biolabs (NEB USER) (Cat. #M5505S, Ipswich, MA).
  • FIGs. 6A and 6B are fluorescent images of urea-polyacrylamide gels showing the results of Example 5 and 6, respectively, which illustrate the site-specific cleavage of the free form (FIGs. 6A (1) and 6B (1), or immobilized form (FIGs. 6A (2) and 6B (2)) of the single-stranded DNA (ssDNA), and the free form (FIGs. 6A (3) and 6B (3)) or immobilized form (FIGs. 6A (4) and 6B (4)) of the double-stranded DNA (dsDNA) by the exemplary enzymes that preferentially or specifically recognize deoxyinosine (I, FIG.
  • S refers to the substrate DNA only;
  • Eco Endo V refers to the in-house prepared E. coli endonuclease V enzyme, which can be referenced to US 2021/0254114A1;
  • Tba Endo V refers to Thermococcus barophilus endonuclease V;
  • Cl refers to the commercial endonuclease V fromNew England Biolabs (NEB Eco EdoV) (Cat.
  • C2 refers to the in-house developed enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and Endo VIII; and C3 refers to an uracilspecific excision reagent from New England Biolabs (NEB USER) (Cat. #M5505S, Ipswich, MA).
  • FIG. 7 is the fluorescent image of urea-polyacrylamide gel showing the results of Example 7, which illustrate the site-specific cleavage of the free form (FIGs. 7(1)) of the single-stranded DNA (ssDNA) and free form (FIGs. 7A (2)) of the double-stranded DNA (dsDNA) by the exemplary enzymes that preferentially or specifically recognize deoxyuridine (U) in the DNA at 37°C.
  • S refers to the substrate DNA only; Eco Endo V refers to the in-house prepared E.
  • coli endonuclease V enzyme which can be referenced to US 2021/0254114A1; Bsu Endo V refers to Bacillus subtilis endonuclease V; Cl refers to the commercial endonuclease V from New England Biolabs (NEB Eco EdoV) (Cat. #M0305S, Ipswich, MA); and C3 refers to an uracil-specific excision reagent from New England Biolabs (NEB USER) (Cat. #M5505S, Ipswich, MA).
  • FIGs. 8A and 8B are fluorescent images of urea-polyacrylamide gels showing the results of Example 8, which illustrate the site-specific cleavage of elongated DNA strand product from the free form (FIGs. 8A (1), 8A (3), 8B (1), and 8B (3)), or the immobilized form of the (FIGs. 8A (2), 8A (4), 8B (2), and 8B (4)) single-stranded DNA (ssDNA) by the exemplary enzymes that preferentially or specifically recognize deoxyinosine (I) (FIGs. 8A (1), 8A (2), 8B (1), and 8B (2)) and deoxyuridine (U) (FIGs.
  • I deoxyinosine
  • U deoxyuridine
  • S refers to the substrate DNA only;
  • Eco Endo V refers to the in-house prepared E. coli endonuclease V enzyme, which can be referenced to US 2021/0254114A1;
  • Pfu Endo V refers to Pyrococcus furiosus endonuclease V;
  • Tba refers to Thermococcus barophilus endonuclease V;
  • Pfu Endo Q refers to Pyrococcus furiosus endonuclease Q;
  • Bpu Endo Q refers to Bacillus pumilus endonuclease Q.
  • the terms “including,” “comprising,” “containing,” and any other variations thereof are intended to cover a non-exclusive inclusion.
  • an object when describing an object “comprises” a limitation, unless otherwise specified, it may additionally include other ingredients, elements, components, structures, regions, parts, devices, systems, steps, or connections, etc., and should not exclude other limitations.
  • any numeral value that falls within the numeral scope herein could be taken as a maximum or minimum value to derive the sub-ranges therefrom.
  • the numeral range “from 10°C to 100°C” comprises any sub-ranges between the minimum value of 10°C to the maximum value of 100°C, such as the sub-ranges from 10°C to 50°C, from 60 °C to 100°C, from 70 °C to 90°C and so on.
  • a plurality of numeral values used herein can be optionally selected as maximum and minimum values to derive numerical ranges.
  • the numerical ranges of 37°C to 55 °C, 37°C to 70°C, and 55 °C to 70°C can be derived from the numeral values of 37°C, 55 °C, and 70°C.
  • the term “about” generally referring to the numerical value meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or ⁇ 0.1% from a given value or range. Such variations in the numerical value may occur by, e.g., the experimental error, the typical error in measuring or handling procedure for making compounds, compositions, concentrates, or formulations, the differences in the source, manufacture, or purity of starting materials or ingredients used in the present disclosure, or like considerations.
  • nucleic acid refers to a guiding nucleotide or ribonucleotide sequence in a single-stranded or double-stranded form, of which the sources are not limited herein, and generally, include naturally occurring nucleotides or artificial chemical mimics.
  • nucleic acid as used herein is interchangeable with the terms including natural or unnatural “oligonucleotide”, “polynucleotide”, “gene”, “cDNA”, “RNA”, and “mRNA”.
  • nucleic acid as used herein also includes nucleic acid analogue.
  • nucleic acid analogue is known to describe compounds or artificial nucleic acids which are functionally or structurally equivalent to naturally existing RNA and DNA.
  • a nucleic acid analogue may have one or more parts of a nucleotide (the phosphate backbone, pentose sugar, and nucleobase) being modified. These modifications on the nucleotide change the structure and geometry of the nucleic acid and its interactions with nucleic acid polymerases.
  • the nucleic acid analogue also encompasses the emerging category of artificial nucleic acids, such as xeno nucleic acids (XNAs), which is designed to have new-to-nature forms of sugar backbone.
  • XNAs xeno nucleic acids
  • nucleic acid analogues include but are not limited to: the universal bases, such as inosine, 3 -nitropyrrole, and 5 -nitroindole, which can form a base-pair with all four canonical bases; the phosphate-sugar backbone analogues, such as peptidenucleic acids (PNA), which affect the backbone properties of the nucleic acid; chemical linker or fluorophore-attached analogues, such as amine-reactive aminoallyl nucleotide, thiol-containing nucleotides, biotin-linked nucleotides, rhodamine-linked nucleotides, and cyanine-linked nucleotides; the fluorescent base analogues, such as 2-aminopurine (2-AP), 3-methylisoxanthopterin (3-MI), 6-methylisoxanthopterin (6-MI), 4-amino-6- methylisoxanthopterin (6-MAP), and 4-d
  • oligonucleotide used herein is generic to any type of polynucleotide including polyribonucleotides, polydeoxyribonucleotides nucleotides, or other polynucleotides that consist of an N-glycoside with a purine or pyrimidine base.
  • oligonucleotide and polynucleotide used herein are not intended to be distinct in length, where these two terms refer only to the molecule structure, and therefore are used interchangeably herein.
  • An oligonucleotide or polynucleotide can further comprise natural, damaged, or modified nucleotides.
  • Nucleic acid bases contained in an oligonucleotide or polynucleotide may be, for example, adenine, thymine, cytosine, guanine, uracil, xanthine, hypoxanthine, isocytosine, isoguanine, 5 -fluorouracil, 5- hydroxymethyluracil, 5 -formylcytosine, 5 -carboxylcytosine, 3 -methyladenine, 3- methylguanine, 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8- dihydroguanine, 5-hydroxylcytosine, 5-hydroxyluracil, dihydroxyuracil, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4- hydroxy-5-N-methylformamidopyrimidine, a formamidopyrim
  • the term “endonuclease activity” used herein refers to an enzymatic activity of breaking the linkage bond at a specific and recognizable nucleic acid site, resulting in a cleaving reaction in a single or double stranded DNA.
  • the endonuclease activity may be provided by naturally occurring enzymes and the modified derivatives thereof.
  • the examples of modified derivatives include enzymatically active mutants/variants, fragments, recombinant proteins derived from the enzymes possessing endonuclease activity.
  • an endonuclease may cleave a single-stranded DNA strand and release the oligonucleotide/polynucleotide with a 5 ’-monophosphate on the one hand and leaves a free 3 ’-hydroxyl group on the remaining nucleic acid strand on the other hand.
  • abasic apurinic/apyrimidinic
  • D-spacer can be interchangeably used to indicate a site at which the base is not present, but the sugar phosphate backbone remains intact. Therefore, the abasic site endonuclease is also known as apurinic/apyrimidinic site endonuclease.
  • the term “template” refers generically to a polynucleotide, or a polynucleotide mimic, which contains the desired or unknown target nucleotide sequence.
  • target sequence the terms “target sequence,” “template polynucleotide,” “target nucleic acid,” “target polynucleotide,” “nucleic acid template,” “template sequence,” and variations thereof, are used interchangeably.
  • template refers to a strand of nucleic acid, on which a complimentary copy is synthesized from nucleotides or nucleotide analogues through the replication of a template-dependent, or template-directed, nucleic acid polymerase.
  • the template strand is, by the convention definition, depicted and described as the “bottom” strand.
  • the non-template strand is often depicted and described as the “top” strand.
  • the “template” strand may also be referred to as the “sense” or “plus” strand and the non-template strand as the “antisense” or “minus” strand.
  • initiator refers to a mononucleoside, a mononucleotide, an oligonucleotide, a polynucleotide, or modified analogues thereof, from which a nucleic acid is to be synthesized by a nucleic acid polymerase de novo.
  • initiator may also refer to an XNA or a peptide nucleic acid (PNA) having a 3 ’-hydroxyl group.
  • primer used herein refers to a short single-stranded oligonucleotide, a polynucleotide, or a modified nucleic acid analogue used in combination with template by a nucleic acid polymerase to initiate nucleic acid synthesis.
  • nucleotide monomer disclosed herein includes canonical nucleotides and nucleotide analogues.
  • nucleotide analogue is known to those skilled in the art to describe the chemically modified nucleotides or artificial nucleotides, which are structural mimics of canonical nucleotides. These nucleotide analogues can serve as substrates for nucleic acid polymerases to synthesize nucleic acid.
  • a nucleotide analogue may have one or more altered components of a nucleotide (e.g., the phosphate backbone, pentose sugar, and nucleobase), which changes the structure and configuration of a nucleotide and affects its interactions with other nucleobases and the nucleic acid polymerases.
  • a nucleotide analogue having altered nucleobase may confer alternative base-pairing and base-stacking properties in the DNA or RNA.
  • the modification at the base may generate various nucleosides such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5 -deoxy uridine, and any other analogues which permits hybridization.
  • modifications may take place at the level of sugar moiety (for example, replacement of a deoxyribose by an analogue), and/or at the level of the phosphate group (for example, boronate, alkylphosphonate, or phosphorothioate derivatives).
  • a nucleotide analogue monomer may have a phosphate group selected from a monophosphate, a diphosphate, a triphosphate, a tetraphosphate, a pentaphosphate, and a hexaphosphate.
  • nucleotide analogues also include nucleotides having a removable blocking moiety.
  • the removable blocking moiety include, but are not limited to, a 3'-O-blocking moiety, a base blocking moiety, and a combination thereof.
  • Examples of the 3'-O-blocking moiety include, but are not limited to, O-azido (O-Ns), O-azidomethyl, O-amino, O-allyl, O-phenoxyacetyl, O-methoxyacetyl, O- acetyl, O-(p-toluene)sulfonate, O-phosphate, O-nitrate, O-[4-methoxy]- tetrahydrothiopyranyl, O-tetrahydrothiopyranyl, O-[5-methyl]-tetrahydrofuranyl, O- [2-methyl, 4-methoxy] -tetrahydropyranyl, O-[5-methyl]-tetrahydropyranyl, and O- tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methyl, and O-acyl.
  • O-azido O-Ns
  • O-azidomethyl O-a
  • Examples of the base blocking moiety may be a reversible dye-terminator.
  • Examples of the reversible dyeterminator include, but are not limited to, a reversible dye-terminator of Illumina MiSeq, a reversible dye-terminator of Illumina HiSeq, a reversible dye-terminator of Illumina Genome Analyzer IIX, a reversible dye-terminator of Helicos Biosciences Heliscope, and a reversible dye-terminator of LaserGen’s Lightning Terminators.
  • polymerase used herein is generically a DNA polymerase including naturally-occurring enzymes and modified derivatives thereof. For example, a sequence modification to remove 5’ to 3’ or 3’ to 5’ exonuclease activity can be applied to a polymerase. Mutations or deletions to functional groups or sequences of a polymerase are also involved to improve the performance of the polymerase.
  • the polymerase may be a template-dependent polymerase or a template-independent polymerase.
  • the polymerase may be selected from the group consisting of a family-A DNA polymerase (e.g., T7 DNA polymerase, Pol I, Pol y, 0, and v), a family-B DNA polymerase (e.g., Pol II, Pol B, Pol Pol a, 6, and s), a family-C DNA polymerase (e.g., Pol III), a family-D DNA polymerase (e.g., PolD), a family-X DNA polymerase (e.g., Pol , Pol o, Pol X, Pol p, and terminal deoxynucleotidyl transferase), a family -Y DNA polymerase (e.g., Pol i, Pol K, Pol r
  • Non-limiting examples of widely employed template-dependent polymerases include T7 DNA polymerase of the phage T7 and T3 DNA polymerase of the phage T3, which are DNA-dependent DNA polymerases; T7 RNA polymerase of the phage T7 and T3 RNA polymerase of the phage T3, which are DNA-dependent RNA polymerases; DNA polymerase I or its fragment known as the Klenow fragment of Escherichia coli, which is a DNA-dependent DNA polymerase; Thermophilus aquaticus DNA polymerase, Tth DNA polymerase and Vent DNA polymerase, which are thermostable DNA-dependent DNA polymerases; eukaryotic DNA polymerase P, which is a DNA-dependent DNA polymerase; telomerase, which is a RNA-dependent DNA polymerase; and non-protein catalytic molecules, such as modified RNA (ribozymes; Unrau & Bartel, 1998) and DNA with template-dependent polymerase activity.
  • Non-limiting examples of the template-independent polymerases include reverse transcriptase, poly A polymerase, DNA polymerase theta (0), DNA polymerase mu (p), DpoIV polymerase, and terminal deoxynucleotidyl transferase. Since polymerases suitable for nucleic acid synthesis, linking nucleotide addition, and nucleic acid synthesis are within the expertise and routine skills of those skilled in the art, further details thereof are omitted herein for the sake of brevity.
  • the method provided herein comprises use of a B-family polymerase.
  • B-family polymerase include, but are not limited to, E. coli DNA polymerase II (Eco), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), Bacillus phage Phi29 DNA polymerase (Phi29), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD), human DNA polymerase delta catalytic p!25 subunit (hPOLD), Sulfolobus solfataricus DNA polymerase (Sso), Pyrobaculum islandicum DNA polymerase (Pis), Thermococcus sp.
  • Esco E. coli DNA polymerase II
  • Pae Pseudomonas aeruginosa DNA polymerase II
  • DNA polymerase (9°N), Thermococcus kodakarensis DNA polymerase (Kodl), Methanococcus maripaludis DNA polymerase (Mma), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus gorgonarius DNA polymerase (Tgo), and Thermococcus litoralis DNA polymerase (Vent).
  • the nucleotide monomer used herein may have a removable blocking moiety.
  • the removable blocking moiety include, but are not limited to, a 3’-O- blocking moiety, a base blocking moiety, and a combination thereof.
  • the nucleotide monomer having a removable blocking moiety is also referred to as a reversible terminator.
  • the nucleotide monomer having the 3 ’-O-blocking moiety is also referred to as a 3 ’-blocked reversible terminator or a 3’-O-modified reversible terminator
  • the nucleotide monomer having a base blocking moiety is also referred to as a 3 ’-unblocked reversible terminator or a 3 ’-OH unblocked reversible terminator.
  • the term “reversible terminator” refers to a chemically modified nucleotide monomer.
  • a reversible terminator When such a reversible terminator is incorporated into a growing nucleic acid by a polymerase, it blocks the further incorporation of another nucleotide monomer by the polymerase.
  • Such “reversible terminator” base and a nucleic acid can be deprotected by chemical or physical treatment, and following such deprotection, the nucleic acid can be further extended by a polymerase.
  • Examples of the 3’-O-blocking moiety include, but are not limited to, O-azidomethyl, O-amino, O-allyl, O- phenoxyacetyl, O-methoxyacetyl, O-acetyl, O-(p-toluene)sulfonate, O-phosphate, O- nitrate, O-[4-methoxy]-tetrahydrothiopyranyl, O-tetrahydrothiopyranyl, O-[5-methyl]- tetrahydrofuranyl, O-[2-methyl, 4-methoxy]-tetrahydropyranyl, O-[5-methyl]- tetrahydropyranyl, and O-tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methylmethyl, and O-acylacyl.
  • Examples of the 3 ’-unblocked reversible terminators include, but are not limited to, 7-[(S)-l-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-7- deaza-dATP, 5-[(S)-l-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl- dCTP, l-[(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]-5-methyl-7-deaza- dGTP, 5-[(S)-l-(5-methoxy-2-nitrophen-yl)-2,2-dimethyl-propyloxy]methyl-dUTP, and 5-[(S)-l-(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-dUTP.
  • the base blocking moiety may also be a reversible dye-terminator.
  • the reversible dye-terminator include, but are not limited to, a reversible dye-terminator of Illumina NovaSeq, a reversible dye-terminator of Illumina NextSeq, a reversible dye-terminator of Illumina MiSeq, a reversible dye-terminator of Illumina HiSeq, a reversible dyeterminator of Illumina Genome Analyzer IIX, a lightning terminator of LaserGen, and a reversible dye-terminator of Helicos Biosciences Heliscope.
  • the guiding nucleotide is a natural, unnatural, or modified nucleotide, wherein the nucleobase of the nucleotide may be, for example, adenine, thymine, cytosine, guanine, uracil, xanthine, hypoxanthine, isocytosine, isoguanine, 5- fluorouracil, 5-hydroxymethyluracil, 5 -formylcytosine, 5-carboxylcytosine, 3- methyladenine, 3-methylguanine, 7-methyladenine, 7-methylguanine, N6- methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxylcytosine, 5-hydroxyluracil, dihydroxyuracil, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6- diamino-4-hydroxy-5-N-methyl formamidopyr
  • the guiding nucleotide contains a nucleobase selected from the group consisting of uracil, xanthine, hypoxanthine, cytosine, and guanine. In some embodiments, the guiding nucleotide contains an apurinic/apyrimidinic lesion, wherein the apurinic/apyrimidinic lesion is an abasic site or a dSpacer.
  • an initiator or primer-template duplex may be used as the starting material for nucleic acid synthesis.
  • the initiator/primer-template duplex may have a 5 ’-end linked to a solid support.
  • the initiator may be directly attached to the solid support or may be attached to the solid support via a linker.
  • solid support examples include, but are not limited to, microarrays, beads (coated or non-coated), columns, optical fibers, wipes, nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, magnetic particles, plastics (e.g., polyethylene, polypropylene, and polystyrene), gel forming materials (e.g., gelatins), lipopolysaccharides, silicates, agarose, polyacrylamides, methyl methracrylate polymers), sol-gels, porous polymers, hydrogels, nanostructured surface nanotubes (e.g., carbon nanotubes), and nanoparticles (e.g., gold nanoparticles or quantum dots).
  • plastics e.g., polyethylene, polypropylene, and polystyrene
  • gel forming materials e.g.,
  • a polymerase may elongate a nucleic acid strand by adding a plurality of nucleotide monomers to the initiator, wherein the nucleotide monomers include a site-specific, recognizable nucleotide monomer comprising a predetermined nucleobase or an apurinic/apyrimidinic lesion, which is defined as a guiding nucleotide.
  • the guiding nucleotide is incorporated in the nucleic acid strand at the designated position, and then a site-specific enzymatic cleavage is performed to retrieve the desired target polynucleotide from the newly synthesized nucleic acid strand.
  • a site-specific enzymatic cleavage is performed to retrieve the desired target polynucleotide from the newly synthesized nucleic acid strand.
  • the desired nucleic acid strands or polynucleotide to be retrieved may contain newly synthesized nucleic acid or may be the commercially available ready-to-use synthetic nucleic acids.
  • the disclosed cleavage enzymes preferentially or specifically cleave at the specific position of the designed nucleic acid strand to release the desired sequence and length of polynucleotide.
  • the present disclosure provides a method of synthesizing a defined sequence and length of polynucleotide enzymatically, wherein the method comprises uses of an initiator having a free 3 ’ -hydroxyl group at the 3 ’ end, a newly synthesized nucleic acid strand by polymerase, and an enzyme/endonuclease having a polynucleotide-cleavage activity, which cleaves a specific phosphodiester bond in the newly synthesized nucleic acid strand.
  • a polymerase incorporates nucleotide monomers containing a guiding nucleotide to the initiator to elongate a nucleic acid strand from the free 3’- hydroxyl group.
  • the guiding nucleotide which is incorporated by polymerase at a specific position of the elongated nucleic acid strand. Thereafter, a selected endonuclease recognizes the guiding nucleotide and cleaves the newly synthesized nucleic acid strand according to the position of the guiding nucleotide to release a the predetermined, or desired, sequence and length of polynucleotide.
  • the selected endonuclease specifically recognizes the position of the guiding nucleotide in the nascent nucleic acid strand and preferably cleaves the second phosphodiester bond 3' to the guiding nucleotide, the first phosphodi ester bond 5' to the guiding nucleotide, the second phosphodiester bond 5' to the guiding nucleotide, or the third phosphodiester bond 5' to the guiding nucleotide, respectively.
  • nucleic acid strand having a free 3 ’-hydroxy group which can be used as a new, or a usable, initiator for another round of nucleic acid synthesis.
  • thermophilic endonuclease can be utilized recognize the position of guiding nucleotide in the nascent nucleic acid strand and to perform a site-specific nucleic acid cleavage. Due to the thermophilic enzyme’s intrinsic thermotolerant property, a thermostable endonuclease may catalyze the nucleic acid cleavage under a wide variety of reaction conditions, such as an elevated reaction temperature.
  • the endonuclease in the present disclosure is an endonuclease V, an endonuclease Q, NucS endonuclease, or EndoMS endonuclease.
  • a method of retrieving a predetermined sequence and length of polynucleotide enzymatically comprises the steps of: providing a synthetic nucleic acids containing a devised guiding nucleotide to be recognized by a selected endonuclease specifically; and subjecting the endonuclease to cleave the synthetic nucleic acids according to the position of the guiding nucleotide in the nucleic acid to release a predetermined, or desired, sequence and length of polynucleotide.
  • the endonuclease specifically recognizes the position of guiding nucleotide in the nucleic acids and preferably cleaves at the second phosphodiester bond 3’ to the guiding nucleotide, the first phosphodiester bond 5' to the guiding nucleotide, the second phosphodiester bond 5' to the guiding nucleotide, or the third phosphodiester bond 5' to the guiding nucleotide, respectively, to obtain the predetermined polynucleotide.
  • the kit may comprise: an initiator having a 3 ’-terminal nucleotide with a free 3 ’-hydroxyl group; a polymerase for incorporating nucleotide monomers , which comprise a guiding nucleotide monomer, to the initiator to elongate a nucleic acid strand from the free 3 ’-hydroxyl group, such that the guiding nucleotide is incorporated in the designated position of newly synthesized nucleic acid strand; and an endonuclease recognizes the guiding nucleotide in the newly synthesized nucleic acid strand and cleave the nucleic acid strand according to the position of the guiding nucleotide to release a the predetermined , or desired, sequence and length of polynucleotide, so that the remaining
  • the kit may comprise: a synthetic polynucleotide having a designated guiding nucleotide; and an endonuclease for recognizing the guiding nucleotide and cleaving the synthetic polynucleotide to release a predetermined, or desired, sequence and length of polynucleotide.
  • the endonuclease recognizes the guiding nucleotide and cleaves at the specific phosphodiester bond according to the position of the guiding nucleotide in the synthetic polynucleotide, so that the predetermined, or desired, sequence and length of polynucleotide is obtained.
  • the properties of site specificity of the endonuclease included in the kits have been described previously.
  • the method and kit provided herein utilizes a Pfu Endo V, Pfu or Bpu Endo Q, Bsu or Tba Endo V, and a nucleic acid initiator or primer/template duplex to generate a nucleic acid strand containing a different, unique guiding nucleotide for a site-specific recognition and the phosphodiester bond cleavage at common enzymatic reaction temperature, such as ambient temperature (e.g., 10°C to 40°C), 37°C, or an elevated reaction temperature, such as 70°C.
  • common enzymatic reaction temperature such as ambient temperature (e.g., 10°C to 40°C), 37°C, or an elevated reaction temperature, such as 70°C.
  • the method and kit provided herein can utilize a nucleic acid initiator or primer/template duplex to generate nucleic acid strand containing an unique guiding nucleotide for recognition and the site-specific nucleic acid cleavage to precisely release the predetermined, or desired, sequence and length of polynucleotide strand at a wide-range of reaction temperatures, which broadens the applications and utilities of enzymatic nucleic acid synthesis.
  • Endonucleases are enzymes that cleave nucleic acid (e.g., DNA or RNA) at phosphodiester bonds connecting the nucleotides and play an important role in maintaining biological functions such as nucleic acid mismatch repairs. Endonucleases are also utilized for applications in DNA manipulation. For example, the T7 endonuclease I is widely used in mismatch repair and genome editing (e.g., mutation and deletion). The enzyme recognizes the DNA mismatch and cleaves the first, second, or third phosphodiester bond 5' to the mismatch position. E. coli Endonuclease V (Eco Endo V) is another enzyme that can specifically recognize DNA position for strand cleavage. The E.
  • E. coli Endo V recognizes the deoxyinosine (di) lesion in DNA and cleaves the second phosphodiester bond at the 3 ’-side of the di lesion and generates a DNA strand break.
  • E. coli Endo V has been used in the de novo enzymatic DNA synthesis to cleave the phosphodiester bond linkage between the initiator and the newly synthesized DNA.
  • the narrow substrate specificity and limited thermal tolerance of E. coli Endo V restricts its utilization and broad applications in emerging enzymatic DNA synthesis.
  • endonucleases such as endonuclease V derived from Pyrococcus furiosus (Pfu) or Thermococcus barophilus (Tba), endonuclease Q derived from Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac) and Bacillus pumilus (Bpu), NucS endonuclease derived from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga), and EndoMS endonuclease from Thermococcus kodakarensis (Tko) have a broader substrate spectrum than E.
  • coli Endo V coli Endo V.
  • endonucleases recognize various deaminated or oxidated bases, such as deoxyuridine or deoxyinosine in nucleic acids.
  • these endonucleases may also recognize apurinic/apyrimidinic lesion in DNA, such as an abasic site or a dSpacer (also known as abasic furan) which is structurally similar to an abasic site.
  • Pfu Endo V, Tba Endo V and Pfu Endo Q, Mac Endo Q, Pab NucS, Tko EndoMS, and Tga NucS can cleave the DNA strand at a much broader reaction temperatures than E. coli Endo V, such as temperatures ranging from 10°C to 100°C, or from 20°C to 30°C, from 30°C to 40°C, from 40°C to 50°C, from 50°C to 60°C, from 60°C to 70°C, from 70°C to 80°C, from 80°C to 90°C, from 90°C to 100°C as demonstrated in the exemplary results.
  • the method and kit disclosed herein provides an improved endonuclease-based nucleic acid strand cleavage approach compared to the conventional art, utilizing a nucleic acid initiator or primer, which includes a different type of deaminated or oxidated bases, such as deoxyuridine or deoxyinosine, for a site-specific recognition and cleavage of nucleic acid strand. Additionally, the method and kit disclosed herein provide an improved thermotolerant endonuclease based nucleic acid cleavage approach that can perform at a wider range of reaction temperatures, which broaden the applications and utilities of enzymatic nucleic acid synthesis.
  • the nucleic acid initiator or primer can be regenerated with a normal 3 ’-hydroxyl group.
  • the nucleic acid initiator, or a primer can be reused for new rounds of enzymatic nucleic acid synthesis.
  • FIGs. 1, 2A, and 2B show the exemplary schemes of the present disclosure in a template-independent (FIG. 1) and a template-directed/template-dependent (FIGs. 2A and 2B) nucleic acid synthesis.
  • an initiator ssDNA
  • the template-directed/template-dependent nucleic acid synthesis requires nucleic acids template attached to the solid support (FIG. 2A) or hybridized to the initiator by annealing method or other known hybridization techniques (FIG. 2B).
  • the primer is used in combination with a template to generate a primer/template duplex (P/T duplex) for synthesizing the DNA.
  • the initiator or primer is elongated by a template-independent or template-dependent polymerase, such as a B-family DNA polymerase, with nucleotide monomers (N) containing a normal 3 ’-hydroxyl group or a 3 ’-blockage chemical moiety to synthesize the oligonucleotide/polynucleotide chain with desired sequence (N-3 to N n ).
  • the sequence of the nucleic acid strand is configured to comprise at least one guiding nucleotide (X) having a predetermined nucleobase or a predetermined apurinic/apyrimidinic (AP) lesion to be recognized by a selected endonuclease.
  • the predetermined nucleobase may be a deoxy uridine or a deoxy inosine
  • the apurinic/apyrimidinic lesion may be an abasic site or a dSpacer, and each recognizable deoxynucleotide residue can be specifically recognized by different types of corresponding endonucleases, respectively.
  • the present method and kit provide an approach to incorporate nucleotide monomers (including canonical and non-canonical nucleotide) to the initiator by the polymerase to synthesize and retrieve a custom-made polynucleotide chain which is site- specifically cleavable according to the needs of the user.
  • nucleotide monomers including canonical and non-canonical nucleotide
  • the inventor has surprisingly discovered that the nucleic acid cleavage activity of the endonucleases disclosed herein can recognize a guiding nucleotide in the sitespecific cleavable region. After sufficient cycles of nucleotide addition/incorporation to generate the desired nucleic acid strand, the endonuclease-based cleavage reaction is introduced to cleave and/or denature the nucleic acid strand or polynucleotide chain that comprises site-specific cleavable region near the guiding nucleotide.
  • the nucleic acid strand having a site-specific cleavable region containing deoxyinosine or deoxyuridine, respectively may be treated with an endonuclease Q derived from Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac), or Bacillus pumilus (Bpu), endonuclease V derived from Bacillus subtilis (Bsu), Escherichia (Eco), Pyrococcus furiosus (Pfu), or Thermococcus barophilus (Tba), NucS endonuclease derived from Thermococcus gammatolerans (Tga) or Pyrococcus a
  • the endonucleases Ei to E4 recognize the guiding nucleotide (X) and cleave phosphodiester bonds between N2 and Ni, between X and N- 1, between N-i, and N-2, and between N-2 and N-3, respectively.
  • X may be deoxy inosine or deoxy uridine;
  • El may be Bsu EndoV, Eco EndoV, Pfu EndoV, or Tba Endo V ;
  • E2 may be Pfu EndoQ, Mac EndoQ, Bpu Endo Q;
  • E3 may be TgaNucS; and E4: may be Tko EndoMS, but the present disclosure is not limited thereto.
  • thermotolerant characteristic of the endonucleases disclosed herein enables a nucleic acid strand cleavage reaction at a wide range of reaction temperatures.
  • present methods of disclosure can be utilized for various conditions and applications of enzymatic nucleic acid synthesis.
  • the site-specific cleavage of the newly synthesized nucleic acid strands or polynucleotide chain by the endonuclease not only releases the desired polynucleotide fragment, but also regenerates a new free 3 ’-hydroxyl group at 3’ end of the remaining nucleic acid strand to be reused for a new round of synthesis reaction, so when combined with a proper design and setting of nucleotide monomer additions, the enzymatic synthesis can be efficiently cycled without interruption of additional enzyme treatment (e.g., dephosphorylation by the phosphatase).
  • additional enzyme treatment e.g., dephosphorylation by the phosphatase
  • the present disclosure provides a precise, efficient, cost-effective, and thermotolerant approach to retrieve the predetermined nucleic acid strand or polynucleotide chain, and to concurrently regenerate the reusable nucleic acid initiator with a free hydroxyl group in one step for accelerating the user-desired nucleic acid synthesis.
  • FAM-45-mer DNA initiator The following synthetic polynucleotide initiator, FAM-45-mer DNA initiator, was used for the template-independent DNA synthesis in this example.
  • This single-stranded 45-mer polynucleotide initiator is modified with a biotin group at the 5 ’-end and an internal fluorescein amidite (FAM) dye at the 23 rd thymidine base (the underline T) and has a free 3 ’ -hydroxyl group at the 3 ’ end thereof.
  • FAM fluorescein amidite
  • the 5’ end of the initiator was immobilized to DynabeadsTM M-280 Streptavidin beads.
  • the initiator may be internally labeledwith a Hexachloro-fluorescein (HEX) or other fluorescent reporter dyes as disclosed herein, and the initiator may be free form or immobilized via 5 ’-end by other solid supports other than DynabeadsTM M-280 Streptavidin beads as disclosed herein.
  • HEX Hexachloro-fluorescein
  • the template-independent nucleic acid synthesis reaction was performed using a B-family DNA polymerase (1 pM) to incorporate a linking 3’ -O-(azidomethyl)- 2’-deoxyuridine triphosphate (100 pM) to the 3’ end of the initiator for 15 minutes.
  • the B-family DNA polymerase (1 pM) was used to stepwise incorporate a 3 ’-O-(azidomethyl)-2’ -deoxy guanosine triphosphate (100 pM) or dNTP mixture (dATP, dCTP, dGTP, and dTTP) (100 pM) to the initiator containing the deoxyuridine (U) at the 3’ terminus.
  • the synthesis reaction was initiated by addition of 10 mM manganese cations and then incubated at 75° C for 15 minutes.
  • reaction was stopped by adding 10 pL of a 2* quench solution (95% deionized formamide and 25 mM EDTA) and subjected to the heat denaturation at 98° C for 10 minutes.
  • the reaction products were analyzed by a 15% denaturing urea-polyacrylamide gel, and the gel results were visualized by Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA., United States).
  • lane S shows the initiator only
  • lane 1 shows that the deoxy uridine triphosphate (dUTP) was efficiently incorporated to the 3 ’-end of the initiator by the B-family DNA polymerase.
  • lanes 2 and 3 illustrate that the B- family DNA polymerase can incorporate deoxyguanosine triphosphate (dGTP) and dNTP (Nl, N2, etc), respectfully, right after the deoxy uridine (U) at the 3’ end of the initiator.
  • the polynucleotide samples of lane 1 and lane 2 have SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the example demonstrates a method for incorporating canonical (e.g., dNTP) or non-canonical nucleotide (e.g., deoxyuridine or deoxy guanosine triphosphate) to the initiator by the polymerase to synthesize a custom-made polynucleotide chain containing at least one exemplary guiding nucleotide located at the desired site to be recognized by the endonuclease according to the needs of the user.
  • canonical e.g., dNTP
  • non-canonical nucleotide e.g., deoxyuridine or deoxy guanosine triphosphate
  • the initiator may also be designed for a template-dependent nucleic acid synthesis in other embodiments and achieve similar results.
  • the complementary template nucleic acids of the initiator may be attached to the solid support (FIG. 2A) or hybridized to the initiator to form a duplex or double strand (FIG. 2B).
  • the single-stranded 38-mer polynucleotides labelled with Hex dye, containing an deoxyuridine (U) or an deoxyinosine (I), designated as Hex-Top-U38-mer and Hex- Top-138-mer, respectively, were synthesized using the method mentioned in Section A above and served as a representative nucleic acid product that contains an initiator and newly synthesized polynucleotide by the template-independent nucleic acid synthesis.
  • the Hex- Top-U38-mer and Hex-Top-I38-mer were hybridized with a complementary singlestranded 38-mer nucleic acid (Bot-A38-mer) at a molar ratio of 1 : 1.5 in the 1 * TE buffer containing 100 mM of NaCl.
  • the DNA annealing reaction was performed in the BioRad thermal cycler machine by heating up the sample mixture to 95°C for 3 minutes and gradually cooling it down (5°C/30 seconds) to 4°C to from a duplex and doublestranded 38-mer nucleic acid.
  • the polynucleotide sequences, buffers, and solutions of the examples are listed in the tables below.
  • the Hex-Top-U38-mer and Hex-Top-I38-mer has a Hexachlorofluorescein (HEX) labeled at the 5’ end thereof and a free 3 ’-hydroxyl group at the 3’ terminus thereof, but the present disclosure is not limited thereto.
  • the 5’ end of the initiator may be labeled by fluorescein amidite (FAM) or other fluorescent reporter dyes disclosed herein.
  • the initiator may be in a free form or immobilized to the solid support disclosed herein in different embodiments.
  • Example 1 Recognition of deoxyinosine and DNA-strand cleavage by Pfu Endo V at two different reaction temperatures
  • the sample groups include (1) only the DNA substrate (S) that serves as a negative control; (2) an in-house E.coli endonuclease V (Eco EndoV) (SEQ ID NO: 7), which can be referred to US 2021/0254114A1; (3) a Pyrococcus furiosus endonuclease V (Pfu Endo V) (SEQ ID NO: 8); (4) an E.coli endonuclease V obtained from New England BioLabs, Ipswich, MA, (Cl); and (5) the enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII (C2) that serves as a positive control for the deoxyinosine excision and the DNA strand cleavage at the guiding nucleotide position in the nucleic acid product thereof.
  • Eco EndoV Eco EndoV
  • sample mixtures (10 l) containing a 100 nM of a single-stranded Hex-Top-I38-mer DNA substrate or a doublestranded Hex-Top-I38-mer/Bot-A38-mer DNA substrate were incubated with 400 nM endonuclease in each sample group in the enzyme reaction buffer.
  • the sample mixtures were incubated at 37°C or 70°C, respectively, for 20 minutes.
  • Each enzyme reaction was stopped by the addition of equal volume (10 pL) of 2x quench solution.
  • the total 20 pL of sample were denatured at 95 °C for 10 min, and 4 pL of each sample mixture were analyzed by 20% denaturing polyacrylamide gel electrophoresis containing 8 M urea in the lx TBE buffer (90 mM Tris-base, 90 mM boric acid, and 2 mM EDTA). The results of the gel were then visualized by Amersham Typhoon scanner (Cytiva, Marlborough, MA).
  • Eco EndoV, Pfu Endo V, and Cl efficiently recognized deoxyinosine (I) and cleaved the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) starting from the deoxyinosine (I) toward 3’ end, or downstream, of the DNA at the reaction temperature of 37°C, thereby releasing a 15-mer single, or double stranded, DNA and the remaining 23-mer single, or double-stranded, DNA with a free 3 ’-hydroxyl group at the terminus, which can readily serve as a new (or a reusable) initiator for the next-round of nucleic acid synthesis.
  • C2 only performed the deoxy inosine (I) excision and DNA strand cleavage at the reaction temperature of 37°C to release 15-mer single, or doublestranded, DNA and failed to generate a free 3 ’-hydroxyl group at the 3’ terminus of the remaining 21-mer single, or double-stranded, DNA, which cannot be used for the new round of nucleic acid synthesis. Also, it was noted that only Pfu Endo V efficiently cleaved or denatured the DNA strand as mentioned above at the reaction temperature of 70°C.
  • Example 2 Deoxyuridine recognition and DNA-strand cleavage by Pfu Endo V at two different reaction temperatures
  • the sample groups include (1) the only DNA substrate (S) that serves as a negative control; (2) an in-house E.coli endonuclease V (Eco EndoV), which can be referred to US 2021/0254114A1; (3) a Pyrococcus furiosus endonuclease V (Pfu Endo V); (4) an E.coli endonuclease V obtained from New England BioLabs, Ipswich, MA, (Cl); and (5) an uracil-specific excision reagent (C3) fromNew England Biolabs (Cat. #M5505S, Ipswich, MA) that serves as a positive control.
  • Eco EndoV in-house E.coli endonuclease V
  • Pfu Endo V Pyrococcus furiosus endonuclease V
  • C3 uracil-specific excision reagent
  • 11) contain a 100 nM of a single-stranded Hex-Top-U38-mer DNA substrate or a double-stranded Hex-Top-U38-mer/Bot-A38-mer DNA substrate.
  • the samples were processed and analyzed as described in Example 1, and details thereof are omitted herein for the sake of brevity.
  • Pfu Endo V efficiently recognized deoxyuridine (U) and cleaved the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) starting from the deoxyuridine (U) toward 3’ end, or downstream, of the DNA at the reaction temperature of 37°C or 70°C, thereby releasing a 15-mer single, or double-stranded, DNA and the remaining 23-mer single, or doublestranded, DNA with a free 3 ’-hydroxyl group at the terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • the sample groups include (1) a Pyrococcus furiosus endonuclease Q (Pfu Endo Q) (SEQ ID NO: 9); (2) an E.coli endonuclease V obtained fromNew England BioLabs, Ipswich, MA, (Cl); and (3) the enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII (C2) that serves as a positive control for the deoxyinosine excision and the DNA strand cleavage at the guiding nucleotide position thereof.
  • Pfu Endo Q Pyrococcus furiosus endonuclease Q
  • V obtained fromNew England BioLabs, Ipswich, MA,
  • Cl the enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII (C2) that serves as a positive control for the deoxyinosine excision and the DNA strand cleavage at the guiding nucleotide position thereof.
  • the sample mixtures (10 l) contain a 100 nM of a single-stranded Hex-Top- 138-mer DNA substrate or a double-stranded Hex-Top-138-mer/Bot- A38-mer DNA substrate.
  • the samples were processed and analyzed as described in Example 1, and details thereof are omitted herein for the sake of brevity.
  • Pfu Endo Q efficiently recognized deoxyinosine (I) and cleaved the phosphodi ester bond between the deoxyinosine (I) and the first nucleotide (C) starting from the deoxy inosine (I) toward 5’ end of the DNA at the reaction temperature of 37°C or 70°C, thereby releasing a 17-mer single or double stranded DNA and the remaining 21-mer single, or double-stranded, DNA with a free 3 ’-hydroxyl group at the 3 ’-terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • Cl can recognized deoxyinosine (I) and cleaved the phosphodiester bond between the first nucleotide (G) and the second nucleotide (G) starting from the deoxy inosine (I) toward 3’ end of the DNA at the reaction temperature of 37°C, thereby releasing a 15-mer single, or doublestranded, DNA and the remaining 23-mer single, or double-stranded, DNA with a free 3 ’-hydroxyl group at the 3 ’-terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • C 1 failed to demonstrate the site-specific cleavage on the DNA at the reaction temperature of 70°C.
  • C2 only performed the deoxyinosine (I) excision and DNA strand cleavage to release 15-mer single, or double-stranded DNA, and failed to generate a free 3 ’-hydroxyl group at the 3’ terminus of the remaining 21-mer single, or double-stranded, DNA, which cannot be used for the new round of nucleic acid synthesis.
  • Example 4 The deoxyuridine recognition and DNA-strand cleavage by Pfu Endo Q at two different reaction temperatures
  • the sample groups include (1) a Pyrococcus furiosus endonuclease Q (Pfu Endo Q); (2) an E.coli endonuclease V obtained from New England BioLabs, Ipswich, MA, (Cl); and (3) an uracil-specific excision reagent (C3) from New England Biolabs (Cat. #M5505S, Ipswich, MA) that serves as a positive control.
  • the sample mixtures (10 pl) contain a 100 nM of a single-stranded Hex-Top-U38-mer DNA substrate or a doublestranded Hex-Top-U38-mer/Bot-A38-mer DNA substrate.
  • the samples were processed and analyzed as described in Example 1, and details thereof are omitted herein for the sake of brevity.
  • Pfu Endo Q efficiently recognized deoxyuridine (U) and cleaved the phosphodiester bond between the deoxyuridine (U) and the first nucleotide (C) starting from the deoxyinosine (I) toward 5 ’ end of the DNA at reaction temperature of 37°C or 70°C, thereby releasing a 17-mer single, or doublestranded, DNA and the remaining 21-mer single, or double-stranded, DNA with a free 3’-hydroxyl group at the 3’-terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • C3 exhibited the similarly sitespecific cleavage on the DNA as Pfu Endo Q to releasel7-mer single, or doublestranded, DNA, while C3 failed to generate a free 3 ’-hydroxyl group at the 3’ terminus of the remaining 21-mer single, or double-stranded, DNA and cannot be used for the new round of nucleic acid synthesis.
  • Cl failed to demonstrate the sitespecific cleavage on the DNA at the reaction temperature of 37 °C or 70°C.
  • Example 5 Deoxy inosine recognition and DNA-strand cleavage by Tba Endo V at two different reaction temperatures
  • the sample groups include (1) only the DNA substrate (S) that serves as a negative control; (2) an in-house E.coli endonuclease V (Eco EndoV), which can be referred to US 2021/0254114A1; (3) a Thermococcus barophilus endonuclease V (Tba Endo V) (SEQ ID NO: 10); (4) the enzyme mixture containing hAAG and EndoVIII (C2) that serves as a positive control for the deoxyinosine excision and the DNA strand cleavage at the guiding nucleotide position thereof.
  • Eco EndoV E.coli endonuclease V
  • Tba Endo V Thermococcus barophilus endonuclease V
  • C2 Thermococcus barophilus endonuclease V
  • the sample mixtures (10 pl) contain a 100 nM of a single-stranded Hex-Top-138-mer DNA substrate or a double-stranded Hex-Top-I38-mer/Bot-A38-mer DNA substrate.
  • the samples were processed and analyzed as described in Example 1, and details thereof are omitted herein for the sake of brevity.
  • Eco EndoV, Tba Endo V, and Cl efficiently recognized deoxyinosine (I) and cleaved the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) starting from the deoxyinosine (I) toward 3’ end of the DNA at the reaction temperature of 37°C, thereby releasing a 15- mer single, or double-stranded, DNA and the remaining 23-mer single, or doublestranded, DNA with a free 3’-hydroxyl group at the 3’-terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • C2 only performed deoxyinosine (I) excision and DNA strand cleavage at the reaction temperature to release 15-mer single, or double-stranded, DNA, and failed to generate a free 3 ’-hydroxyl group at the 3’ terminus of the remaining 21-mer single, or double-stranded, DNA and cannot be used for the new round of nucleic acid synthesis.
  • Tba Endo V efficiently cleaved, or denatured, the DNA strand as mentioned above at the reaction temperature of 70°C as compared with Eco EndoV and Cl.
  • Example 6 Deoxy uridine recognition and DNA-strand cleavage by Tba Endo V at two different reaction temperatures
  • the sample groups include (1) only the DNA substrate (S) that serves as a negative control; (2) an in-house E.coli endonuclease V (Eco EndoV), which can be referred to US 2021/0254114A1; (3) a Thermococcus barophilus endonuclease V (Tba Endo V); (4) an uracil-specific excision reagent (C3) fromNew England Biolabs (Cat. #M5505S, Ipswich, MA) that serves as a positive control.
  • Eco EndoV E.coli endonuclease V
  • Tba Endo V Thermococcus barophilus endonuclease V
  • C3 uracil-specific excision reagent
  • the sample mixtures (10 pl) contain a 100 nM of a single-stranded Hex-Top-U38-mer DNA substrate or a double-stranded Hex-Top-U38-mer/Bot-A38-mer DNA substrate.
  • the samples were processed and analyzed as described in Example 1, and details thereof are omitted herein for the sake of brevity.
  • Tba Endo V efficiently recognized deoxyuridine (U) and cleaved the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) starting from the deoxy inosine (I) toward 3’ end of the DNA at reaction temperature of 70°C, thereby releasing a 15-mer single, or double stranded, DNA and the remaining 23-mer single, or double-stranded, DNA with a free 3’-hydroxyl group at the 3’-terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • Tba Endo V shares comparable experimental results as Pfu Endo V and Q. As shown in the Examples 5 and 6 (referring to FIGs. 6A and 6B, respectively), Tba Endo
  • E. coli Endo V efficiently and site-specifically cleaved the DNA strand from a DNA initiator/primer containing either a deoxyinosine or a deoxyuridine.
  • E. coli Endo V only cleaves the DNA strand from a DNA initiator/primer containing a deoxyinosine.
  • Tba Endo V efficiently and site-specifically cleaves the DNA strand from a DNA initiator/primer containing either a deoxyinosine or a deoxyuridine at both 37°C and
  • Pfu Endo V, Pfu Endo Q, and Tba Endo V efficiently and site-specifically cleaved the DNA strand of the synthesized single, or double-stranded, DNA containing either a dexoyinosine or a dexoyuridine.
  • Eco Endo V could only cleave the DNA strand from the DNA containing the dexoyinosine.
  • Pfu Endo V, Pfu Endo Q, and Tba Endo V efficiently and site-specifically cleaves the DNA at both 37°C and 70°C, while the E.
  • coli Endo V only cleaves the DNA strand at 37°C. Additionally, the free form or immobilized form of the initiator did not affect the DNA cleavage activity of Pfu Endo V, Pfu Endo Q, and Tba Endo V. Also, although the positive control C2 and C3 could both cleave the DNA, they failed to generate a free 3 ’-hydroxyl group at the 3’ terminus of the remaining single, or double-stranded, DNA and cannot be used for the new round of nucleic acid synthesis.
  • Pfu Endo V, Pfu Endo Q, and Tba Endo V can site-specifically recognize a guiding nucleotide, efficiently cleave the nucleic acid strand according to the position of the guiding nucleotide, and effectively regenerate an initiator with a free 3 ’-hydroxyl group at the 3 ’-terminus at a wide range of reaction temperatures including hyperthermal reaction temperatures.
  • Example 7 Deoxyuridine recognition and DNA-strand cleavage by Bsu Endo V
  • the sample groups include (1) only the DNA substrate (S) that serves as a negative control; (2) an in-house E.coli endonuclease V (Eco EndoV), which can be referred to US 2021/0254114A1; (3) a Bacillus subtilis endonuclease V (Bsu Endo V) (SEQ ID NO: 11); (4) an E.coli endonuclease V obtained from New England BioLabs, Ipswich, MA, (Cl); and (5) an uracil-specific excision reagent (C3) from New England Biolabs (Cat. #M5505S, Ipswich, MA) that serves as a positive control.
  • Eco EndoV E.coli endonuclease V
  • Bsu Endo V Bacillus subtilis endonuclease V
  • C3 uracil-specific excision reagent
  • the sample mixtures (10 pl) contain a 100 nM of a single-stranded Hex-Top-U38-mer DNA substrate or a double-stranded Hex-Top-U38-mer/Bot- A38-mer DNA substrate.
  • the samples were processed and analyzed as described in Example 1, and details thereof are omitted herein for the sake of brevity.
  • Bsu Endo V efficiently recognized deoxyuridine (U) and cleaved the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) starting from the deoxyuridine (U) toward 3’ end of the DNA, thereby releasing a 15-mer single, or double-stranded, DNA and the remaining 23-mer single, or double-stranded, DNA with a free 3’-hydroxyl group at the 3’- terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • Eco EndoV and Cl failed to demonstrate such sitespecific cleavage or denaturation.
  • C3 only cleaved the phosphodiester bond between the deoxyuridine (U) and the first nucleotide (G) starting from the deoxyuridine (U) toward 5’ end of the DNA to release a 17-mer single, or doublestranded, DNA and failed to generate a free 3 ’-hydroxyl group at the 3’ terminus of the remaining 21-mer single, or double-stranded, DNA and cannot be used for the new round of nucleic acid synthesis.
  • Bsu Endo V can site-specifically recognize a guiding nucleotide, efficiently cleave the nucleic acid strand according to the position of the guiding nucleotide, and effectively regenerate an initiator with a free 3 ’ -hydroxyl group at the 3 ’-terminus.
  • Example 8 Deoxy inosine and deoxy uridine recognition and DNA-strand cleavage by Endo V and Endo Q at three different reaction temperatures
  • the sample groups include (1) only the DNA substrate (S) that serves as a negative control; (2) an in-house E.coli endonuclease V (Eco EndoV), which can be referred to US 2021/0254114A1; (3) a Pyrococcus furiosus endonuclease V (Pfu Endo V); (4) a Pyrococcus furiosus endonuclease Q (Pfu Endo Q); (5) a Thermococcus barophilus endonuclease V (Tba Endo V); and (6) a Bacillus pumilus endonuclease Q (Bpu Endo Q) (SEQ ID NO: 12).
  • sample mixture (10 ul) containing a 100 nM of a single-stranded Hex-Top-U38mer DNA substrate or Hex-Top-138mer DNA substrate were incubated with 200 nM endonuclease of each sample group in the enzyme reaction buffer.
  • the sample mixtures were incubated at 37°C, 55°C, or 60°C, respectively, for 20 minutes.
  • Each enzyme reaction mixture was stopped by the addition of equal volume (10 pL) of2x quench solution.
  • the total 20 pL of sample were denatured at 95°C for 10 min, and 4 pL of each sample mixture were analyzed by 20% denaturing polyacrylamide gel electrophoresis containing 8 M urea in the lx TBE buffer (90 mM Tris-base, 90 mM boric acid, and 2 mM EDTA). The results of the gel were then visualized by Amersham Typhoon scanner (Cytiva, Marlborough, MA).
  • Pfu Endo V and Tba Endo V efficiently recognize deoxyinosine (I) or deoxyuridine (U) and cleaves the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) starting from the deoxy inosine (I) or deoxy uridine (U) toward 3’ end of the DNA, thereby releasing a 15-mer single, or double-stranded, DNA and the remaining 23-mer single, or double-stranded, DNA with a free 3 ’-hydroxyl group at the 3 ’-terminus, which can readily serve as a new, or reusable, initiator for the next-round of nucleic acid synthesis.
  • both Pfu Endo Q and Bpu Endo Q efficiently recognize deoxy inosine (I) or deoxy uridine (U) and cleave the phosphodi ester bond between the deoxyinosine (I), or deoxyuridine (U), and the first nucleotide (C) starting from the deoxy inosine (I), or deoxy uridine (U), toward the 5’ end of the DNA at the reaction temperature, thereby releasing a 17-mer single, or double-stranded, DNA and the remaining 21-mer single, or double-stranded, DNA with a free 3’-hydroxyl group at the 3 ’-terminus, which can readily serve as a new, or reusable, initiator for the nextround of nucleic acid synthesis.
  • the E. coli Endo V only cleaves the DNA strand containing a deoxyuridine at 37°C.
  • Pfu Endo V, Pfu Endo Q, Tba Endo V, and Bpu Endo Q can site-specifically recognize a guiding nucleotide, efficiently cleave the nucleic acid strand according to the position of the guiding nucleotide, and effectively regenerate an initiator with a free 3’-hydroxyl group at the 3’-terminusat a wide range of reaction temperatures including ambient reaction temperatures.

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Abstract

L'invention concerne un procédé et un kit de synthèse et de récupération d'un polynucléotide par voie enzymatique, comprenant un initiateur contenant un groupe 3'-hydroxyle libre; une polymérase pour incorporer des monomères nucléotidiques dans l'initiateur pour former un brin d'acide nucléique à partir du groupe 3'-hydroxyle libre, le brin d'acide nucléique comprenant un nucléotide de guidage à reconnaître par une endonucléase; et l'endonucléase clivant le brin d'acide nucléique pour libérer un polynucléotide prédéterminé et nouvellement former un groupe 3'-hydroxyle libre à l'extrémité 3' du brin d'acide nucléique restant qui sert d'initiateur réutilisable. L'invention concerne également un kit de synthèse et de récupération d'un polynucléotide à l'aide du procédé.
PCT/US2023/060847 2022-01-28 2023-01-18 Synthèse enzymatique de polynucléotide WO2023147239A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055742A1 (en) * 2006-07-26 2010-03-04 Nishikawa Rubber Co., Ltd. Method for amplification of nucleotide sequence
US20160304845A1 (en) * 2013-05-10 2016-10-20 Kyushu University, National University Corporation New dna cleavage enzyme
US20210189447A1 (en) * 2019-12-23 2021-06-24 Cheng-Yao Chen Method and kit for template-independent nucleic acid synthesis
WO2021178809A1 (fr) * 2020-03-06 2021-09-10 Life Technologies Corporation Synthèse et assemblage d'acide nucléique à fidélité de séquence élevée

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055742A1 (en) * 2006-07-26 2010-03-04 Nishikawa Rubber Co., Ltd. Method for amplification of nucleotide sequence
US20160304845A1 (en) * 2013-05-10 2016-10-20 Kyushu University, National University Corporation New dna cleavage enzyme
US20210189447A1 (en) * 2019-12-23 2021-06-24 Cheng-Yao Chen Method and kit for template-independent nucleic acid synthesis
WO2021178809A1 (fr) * 2020-03-06 2021-09-10 Life Technologies Corporation Synthèse et assemblage d'acide nucléique à fidélité de séquence élevée

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